The phagocyte respiratory burst enzyme, NADPH oxidase, produces superoxide anion. The precursor for other oxygen species (H202, hypochiorous acid) that play important roles in host defense and inflammation and/or act as intracellular signaling molecules. Our long-term goal is to elucidate the signaling pathways and biochemical mechanisms leading to NADPH oxidase activation. Studies in intact cells suggest that the lipid second messengers resulting from the receptor-initiated activation of phospholipase D (PLD) regulate the activation of the respiratory burst. PLD activation increases intracellular levels of phosphatidic acid (PA), which can be converted by the cell to diacyiglycerol (DG). Both PA and DG act as second messengers, but the protein targets involved in NADPH oxidase activation are unclear. DG activates protein kinase C (PKC), which is strongly linked to NADPH oxidase activation. Our results suggest that PA and DG regulate NADPH oxidase activation by indirect and direct mechanisms. The indirect mechanisms involve activation of PKC isoforms and a novel protein kinase (PAPK). These protein kinases phosphorylate the oxidase components p47phax and p22phax and enhance oxidase activation in cell-free systems. The direct mechanisms involve interaction of PA and DG with oxidase component(s). These lipids induce NADPH oxidase activation in a cell-free system when only oxidase proteins are present, hence phosphorylation is not possible. Furthermore, PA binds to p47phax, identifying this protein as a potential functional target of PA. We propose that these indirect (phosphorylation-dependent) and direct (binding of PA and DG to oxidase components) mechanisms mediate PLD-initiated NADPH oxidase activation. The following specific aims are designed to test this hypothesis and to elucidate the biochemical mechanisms involved. In Aim 1, we will test whether PLD-initiated NADPH oxidase activation involves synergy between indirect phosphorylation-dependent and direct lipid-induced mechanisms. We will use several approaches (identification of PKC/PAPK isoforms regulated by PLD, selective reduction of PKC/PAPK expression with antisense oligonucleotides, functional effects of phosphorylation site mutants of p47phax) In Aim 2, we will test whether direct interaction of PA and DG with oxidase proteins is required for PLD initiated NADPH oxidase activation. We will mutate the PA-binding sites in p47phax and characterize the effects of PA binding on p47 function and oxidase activation. Similarly, we will identify the oxidase component(s) that bind(s) DG and assess the role of DG-binding in NADPH oxidase activation. These studies should provide new insights into the regulation of NADPH oxidase activation.